AIveolar hypoxia is observed in many physiological and pathological conditions including chronic obstructive pulmonary disease, cardiogenic pulmonary edema, acute respiratory distress syndrome and ascent to high altitude. The effect(s) of hypoxia on the function and cytoskeleton of the alveolar epithelium, however, have yet to be elucidated. The cytoskeleton is largely responsible for a cell s structural support, and keratin intermediate filaments (IFs) are known to play an important role in maintaining the integrity of epithelial cells. In alveolar epithelial cells (AEC) keratin IFs are the major structural proteins. The importance of an intact keratin IF network for alveolar epithelial function is demonstrated by our finding of profoundly impaired alveolar fluid clearance in keratin 8-deficient mice. This proposal is focused on determining whether hypoxia-induced changes in the keratin IF network leads to AEC dysfunction. We hypothesize that hypoxia generates mitochondrial reactive oxygen species (ROS) in AEC, which activate protein kinases that phosphorylate keratin proteins and regulate the organization, disassembly and degradation of the keratin IF. Modifications to keratin IFs in the alveolar epithelial cell may contribute to impaired alveolar epithelial function. We have formulated four interrelated specific aims to study the hypoxia-induced regulation of keratin IFs in the alveolar epithelium. Specific Aim 1. To determine whether hypoxia, via mitochondrial reactive oxygen species, affects the assembly state of keratin intermediate filament network in alveolar epithelial cells. Specific Aim 2. To determine whether hypoxiainduced changes in keratin IFs are regulated by protein kinase C-dependent phosphorylation of keratin 8 and 18 in alveolar epithelial cells. Specific Aim 3. To determine whether hypoxia-induces the degradation of keratin 8 and 18 via the ubiqutin-proteasome pathway. Specific Aim 4. To determine whether hypoxiainduced changes in keratin IFs modulate alveolar epithelial function in keratin 8 knockout mice. The proposed experiments will determine the molecular mechanisms that regulate the hypoxia mediated reorganization and/or disassembly of keratin IFs. The consequences of this reorganization on alveolar epithelial function will be examined both in vitro and in vivo using AEC, rats and K8-deficient mice. Completion of the proposed studies will provide novel insights on the role of keratin IF in the pathogenesis of hypoxia-induced alveolar epithelial dysfunction, which is of biological and physiological importance in patients with pulmonary edema.